A fluorogenic peptide containing the processing site of human SARS corona virus S-protein:: Kinetic evaluation and NMR structure elucidation

Human severe acute respiratory syndrome coronovirus (hSARS-CoV) is the causative agent for SARS infection. its surface glycoprotein (spike protein) is considered to be one of the prime targets for SARS therapeutics and intervention because its proteolytic maturation by a host protease is crucial for host-virus fusion. Using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS-CoV spike protein (Abz-(755)Glu-Gln-Asp-Arg-Asn-Thr-Arg-Glu-Val-Phe-Ala-Gln(766)-Tyx-NH2) and in vitro studies, we show that besides furin, other PCs, like PC5 and PC7, might also be involved in this cleavage event. Through kinetic measurements I with recombinant PCs, we observed that the peptide was cleaved efficiently by both furin and PC5, but very poorly by PC7. The cleavage could be blocked by a PC-inhibitor, alpha 1-PDX, in a dose-dependent manner. Circular dichroism spectra indicated that this peptide possesses a high degree of sheet structure. Following cleavage by furin, the sheet content increased, possibly at the expense of turn and random structures. (HNMR)-H-1 spectra from 2D COSY and ROESY experiments under physiological buffer and pH conditions indicated that this peptide possesses a structure with a turn at its C-terminal segment, close to the cleavage site. The data suggest that the cleavable peptide bond is located within the most exposed domain; this is supported by the nearby turn structure. Several strong to weak NMR ROESY correlations were detected, and a 3D structure of the spike IQF peptide that contains the crucial cleavage site R-761 down arrow E has been proposed.